Download Enzyme Mechanisms

Enzyme Mechanisms
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Oxidoreductases catalyze oxidation-reduction reactions.
Transferases catalyze transfer of functional groups from one
molecule to another.
Hydrolases catalyze hydrolytic cleavage.
Lyases catalyze removal of a group from or addition of a group to a
double bond, or other cleavages involving electron rearrangement.
Isomerases catalyze intramolecular rearrangement.
Ligases catalyze reactions in which two molecules are joined.
Types of Enzymes
Two Models for Enzyme-Substrate
Interaction
Induced Conformational Change in
Hexokinase
Coenzymes
Example of a Coenzyme Involved in
Oxidation/Reduction Reactions
Hydride (H:-) transfer
(nicotinamide adenine dinucleotide)
Stereospecificity of Yeast Alcohol Dehydrogenase
ethanol
acetaldehyde
pro-S
pro-R
pro-R
pro-S
Vennesland and
Westheimer, 1950
Stereospecificity Conferred by an Enzyme
Catalytic Mechanisms
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Acid-base catalysis
Covalent catalysis
Metal ion catalysis
Electrostatic catalysis
Proximity and orientation effects
Preferential binding to transition state
(transition state stabilization)
Acid-Base Catalysis
Keto-Enol Tautomerism:
Uncatalyzed vs. Acid- or Base-Catalyzed
Example of Acid-Base Catalysis: Bovine
Pancreatic RNase A
Covalent Catalysis: Nucleophiles and
Electrophiles
Protonated
Example of Covalent Catalysis:
Decarboxylation of Acetoacetate
Lysine side chain e-amino group on
enzyme is nucleophile in attack on
substrate.
Electrophilic “electron sink”
Example of Metal Ion Catalysis:
Carboxypeptidase A
Example of Metal Ion Catalysis: Carbonic
Anhydrase
Carbonic anhydrase catalyzes
the reaction:
CO2 + H2O HCO3− + H+

Enolase Mechanism
Entropic and Enthalpic Factors in
Catalysis
Proximity and
orientation
effects
Transition state
stabilization through
preferential binding of
transition state
Proximity and Orientation Effects
Enzymes Are Complementary to
Transition State
Serine Protease Mechanism: Multiple
Catalytic Mechanisms at Work
Structure of the Serine Protease
Chymotrypsin
Serine Protease Substrate Specificity and
Active-Site Pockets
Trypsin cleaves
amide bond
immediately Cterminal to basic
amino acid
residues.
Substrate specificity in serine proteases through
active-site binding of side chain of amino acid
residue adjacent to amide bond that will be
cleaved.
Chymotrypsin
cleaves amide
bond immediately
C-terminal to
hydrophobic amino
acid residues.
Serine Nucleophile in Serine Proteases
The Pre-Steady State in ChymotrypsinCatalyzed Hydrolysis of p-Nitrophenyl
Acetate
P1
k1
H 2O
E+S
ES  EP2  E + P2

k
k
k
-1
vo = kcat[E]t[S]/(KM + [S])
Steady-state velocity, where
kcat = k2k3/(k2 + k3)
KM = KSk3/(k2 + k3)
KS = k-1/k1
For chymotrypsin with ester
substrates: k2 >> k3
Release of P1 faster than EP2
breaks down to E + P2
2
3
Serine Protease Mechanism
Catalytic triad:
•Acid-base catalysis
•Covalent catalysis
•Proximity/orientation
effects
•Also (not depicted here)
- electrostatic catalysis
and transition state
stabilization
carboxylic acid,
The Oxyanion Hole In Serine Proteases
Role of oxyanion hole in serine protease mechanism:
•Electrostatic catalysis
•Preferential binding of transition state
Trypsin/Bovine Pancreatic Trypsin
Inhibitor (BPTI) Complex
Trypsin-BPTI complex
resembles tetrahedral
transition state.
Transition State in Proline Racemase Reaction
and Transition State Analogs
Proline racemase preferentially binds transition state, stabilizing it, and is
potently inhibited by transition state analogs.
RNA-Based Catalysts (Ribozymes)
Cleavage of a Typical Pre-tRNA by
Ribonuclease P
Ribonuclease P is a ribonucleoprotein
(RNA- and protein-containing complex),
and the catalytic component is RNA.
An even more complex example of an
RNA- and protein-containing enzyme
system is the ribosome. The central
catalytic activity of the ribosome (peptide
bond formation) is catalyzed by an RNA
component.
tRNA substrate of
ribonuclease P
Catalysis by the Intervening Sequence in
Tetrahymena Preribosomal RNA
RNA by itself without
any protein can be
catalytic.
Enzyme Regulation
Effect of Cooperative Substrate Binding
on Enzyme Kinetics
Cooperative enzymes do not obey simple Michaelis-Menten kinetics.
Effect of Extreme Homoallostery
Homotropic allosteric
regulation by
substrate: S at one
active site affects
catalysis of S P at
 sites on enzyme
other
complex.
Extreme positive
cooperativity
depicted here
[S]c = critical substrate concentration

Heteroallosteric Control of an Enzyme
Heterotropic allosteric
regulation: non-S
effectors modulate
catalysis of S P.
(positive
allosteric
effector)


(negative
allosteric
effector)
A Model for Enzyme Regulation: Aspartate
Transcarbamoylase (Aspartate
Carbamoyltransferase) in Pyrimidine
Synthesis
Aspartate Transcarbamoylase (ATCase)Catalyzed Reaction
Feedback Inhibition of ATCase by CTP
Regulation of ATCase by ATP and CTP
ATP is a positive heterotropic
allosteric effector of ATCase,
while CTP is a negative
heterotropic allosteric effector.
Detailed Structure of One Catalytic
Subunit and Adjacent Regulatory Subunit
of ATCase
Quaternary Structure of ATCase in T
State and R State
CTP and ATP bind at regulatory site, but CTP preferentially binds
in T state, while ATP preferentially binds R state.
X-Ray Structure of Aspartate
Transcarbamoylase
T State
T State
“top” view
“side” view
R State
“side” view